Pleiotrophin-Loaded Mesoporous Silica Nanoparticles as a Possible Treatment for Osteoporosis

Osteoporosis is the most common type of bone disease. Conventional treatments are based on the use of antiresorptive drugs and/or anabolic agents. However, these treatments have certain limitations, such as a lack of bioavailability or toxicity in non-specific tissues. In this regard, pleiotrophin (PTN) is a protein with potent mitogenic, angiogenic, and chemotactic activity, with implications in tissue repair. On the other hand, mesoporous silica nanoparticles (MSNs) have proven to be an effective inorganic drug-delivery system for biomedical applications. In addition, the surface anchoring of cationic polymers, such as polyethylenimine (PEI), allows for greater cell internalization, increasing treatment efficacy. In order to load and release the PTN to improve its effectiveness, MSNs were successfully internalized in MC3T3-E1 mouse pre-osteoblastic cells and human mesenchymal stem cells. PTN-loaded MSNs significantly increased the viability, mineralization, and gene expression of alkaline phosphatase and Runx2 in comparison with the PTN alone in both cell lines, evidencing its positive effect on osteogenesis and osteoblast differentiation. This proof of concept demonstrates that MSN can take up and release PTN, developing a potent osteogenic and differentiating action in vitro in the absence of an osteogenic differentiation-promoting medium, presenting itself as a possible treatment to improve bone-regeneration and osteoporosis scenarios.


Introduction
The current population is ageing because life expectancy has increased in the last decades [1]. As a consequence, the impact of bone diseases on society is increasing, especially in postmenopausal women and in the elderly [1,2]. In this context, osteoporosis is becoming a more common disease in the ageing population with altered bone remodeling, where the osteoblasts (bone-forming cells) and osteoclasts (bone-resorbing cells) work in an uncoordinated process [3]. The lack of cellular communication leads to the typical loss of bone mass together with the deterioration of the microarchitecture of bone tissue and the consequent risk of fracture. It affects one in three women and one in five men according to the International Osteoporosis Foundation (IOF) and is associated with more than nine million fractures worldwide [4]. As a result, the disease has both a real human and socio-economic impact.
Current treatments for osteoporosis are based on the use of antiresorptive drugs, which inhibit the action of osteoclasts, and/or anabolic agents that activate the action of Scheme 1. Illustration of the preparation, PEI pre-coating, pleiotrophin (PTN) loading, and in lular release of MSN@PEI-PTN nanocarriers and their effects in human Mesenchymal Stem (hMSCs) and mouse pre-osteoblastic MC3T3-E1 cells.

Synthesis of Mesoporous Silica Nanoparticles
The synthesis of MCM-41 type MSNs was carried out using a modified S method [37]. Briefly, 0.6 g of cetyl-trimethylammonium bromide (CTAB) and 300 m 0.32 M ammonium hydroxide solution was added to in a 500 mL beaker. The mixtur carried out at 50 °C for 1 h, under moderate stirring. Then, 6 mL of a 1:4 ratio of tetra orthosilicate (TEOS) and ethanol solution was added and left to stand overnight. the suspension mixture was pipetted, and the precipitate formed was discarded. Th pension was subjected to a hydrothermal-ageing treatment at 70 °C in a closed bottle night, and the solid was separated by centrifugation. The surfactant used as a templa the mesoporous structure was removed by an ion-exchange extraction process, wi extraction NH4NO3 (10 mg/mL) solution in ethanol (95%) for two cycles, to ensure mum surfactant extraction. These MSNs were synthesized by a co-condensation of with a functional organosilane 3-trihydroxysilylpropyl methylphosphonate TS (50%w, 0.14 mL, 0.37 mmol). In addition, MSNs were labelled by a co-condensati amino propyltriethoxysilane APTES (98%) with Rhodamine-B or fluorescein isocy (FITC, 1.54 × 10 −3 mmol).

Poly(ethylenimine) Grafting
To perform the polymer coating, 5 mg of phosphonate-modified MSNs wer persed in 1 mL of absolute ethanol with a (2:1) MSN@PEI (5KDa) ratio (2:1), show previous studies to be non-toxic [31,34,36]. Although some cytotoxicity has been re edly associated with this type of polymer, below a certain molecular weight, it is per biocompatible [31,32,36]. The mixture, well-dispersed in ultrasound, was stirred f min at room temperature and then washed with phosphate-buffered saline and eth All reagents used for the synthesis of MSNs and their coating were commercial pro from Merck (Spain) and were used without further purification. Scheme 1. Illustration of the preparation, PEI pre-coating, pleiotrophin (PTN) loading, and intracellular release of MSN@PEI-PTN nanocarriers and their effects in human Mesenchymal Stem Cells (hMSCs) and mouse pre-osteoblastic MC3T3-E1 cells.

Synthesis of Mesoporous Silica Nanoparticles
The synthesis of MCM-41 type MSNs was carried out using a modified Stöber method [37]. Briefly, 0.6 g of cetyl-trimethylammonium bromide (CTAB) and 300 mL of a 0.32 M ammonium hydroxide solution was added to in a 500 mL beaker. The mixture was carried out at 50 • C for 1 h, under moderate stirring. Then, 6 mL of a 1:4 ratio of tetraethyl orthosilicate (TEOS) and ethanol solution was added and left to stand overnight. Then, the suspension mixture was pipetted, and the precipitate formed was discarded. The suspension was subjected to a hydrothermal-ageing treatment at 70 • C in a closed bottle overnight, and the solid was separated by centrifugation. The surfactant used as a template for the mesoporous structure was removed by an ion-exchange extraction process, with an extraction NH 4 NO 3 (10 mg/mL) solution in ethanol (95%) for two cycles, to ensure maximum surfactant extraction. These MSNs were synthesized by a co-condensation of TEOS with a functional organosilane 3-trihydroxysilylpropyl methylphosphonate TSPMP (50%w, 0.14 mL, 0.37 mmol). In addition, MSNs were labelled by a co-condensation of amino propyltriethoxysilane APTES (98%) with Rhodamine-B or fluorescein isocyanate (FITC, 1.54 × 10 −3 mmol).

Poly(ethylenimine) Grafting
To perform the polymer coating, 5 mg of phosphonate-modified MSNs were dispersed in 1 mL of absolute ethanol with a (2:1) MSN@PEI (5KDa) ratio (2:1), shown in previous studies to be non-toxic [31,34,36]. Although some cytotoxicity has been reportedly associated with this type of polymer, below a certain molecular weight, it is perfectly biocompatible [31,32,36]. The mixture, well-dispersed in ultrasound, was stirred for 30 min at room temperature and then washed with phosphate-buffered saline and ethanol. All reagents used for the synthesis of MSNs and their coating were commercial products from Merck (Spain) and were used without further purification.

Physicochemical Characterization of MSNs
The MSNs were characterized by electronic microscopy and chemical-and surfacecharacterization techniques. The micrographs were obtained by transmission-electron microscopy (TEM) in the JEOL JEM 2100 equipment at 200 kV, which was equipped with a KeenView camera. Particle size was determined by Dynamic Light Scattering (DLS) analyzed together with Z-potential (ζ) on a Zetasizer Nano ZS (Malvern Instruments, Westborough, MA, USA) equipped with a 633 nm red laser, which was measured in a disposable plastic cell in water at room temperature. For the analysis of surface and textural characteristics, a Micromeritics 3 Flex (Norcross, GA, USA) was used. For these measurements, the MSN samples were previously degassed for 24 h at 120 • C under vacuum. Surface area (S BET ) and pore volume (Vp) were calculated using the Brunauer-Emmett-Teller (BET) method, and pore-size distributions were obtained using a Density Functional Theory (DFT) method and a hydroxylated cylindrical pore-surface model. According to the literature, the accuracy of the DFT method is higher than with BJH theory and offers a more reliable fit at pore sizes smaller than 20 nm, with an ability to detect the microporosity of the structure. Diffractograms were obtained by small-angle X-ray diffraction (SA-XRD) on a Philips X-Pert MPD diffractometer equipped with Cu-Kα radiation. This technique revealed the arrangement of the mesostructure of the pores inside the MSNs, analyzed in a low-angle range of 2θ (0.5-10 • ). Thermogravimetric analysis of MSNs was carried out to measure extraction efficiency, quantify the degree of MSN@PEI coating, and assess PTN-protein loading, which was carried out by quantifying the mass loss in a fixed range between 150 and 600 • C. The data obtained using a PerkinElmer Pyris Diamond TG/DTA were obtained with increasing temperature conditions from 5 • C/min up to 600 • C. Chemical characterizations by Fourier-transform infrared spectroscopy (FTIR) were carried out using a Nicolet Nexus spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) that was equipped with a SMART Golden Gate diffuse reflectance accessory for attenuated total reflection (ATR) analysis.

Cell Types
Cell assays were performed using MC3T3-E1 mice pre-osteoblastic cells (ECACC 99072810) and human mesenchymal stem cells (hMSCs) isolated from normal (non-diabetic) adult human bone marrow extracted from bilateral punctures of the posterior iliac crests of healthy volunteers (Lonza, PT-2501, Batch Nº: 19TL16885) [38,39]. The cells were maintained at 37 • C, 5% CO 2 , and 95% humidity in alfa MEM medium (supplemented with 10% FBS, L-glutamine, and antibiotic) and specialized mesenchymal stem-cell culture medium (supplemented with SingleQuots ® (Poietics TM )), respectively. The cells used at passages #5-6 were seeded for assays at a density of 2.5 × 10 4 cells/cm 2 . When they reached 40-60% confluence, they were incubated for 2 h with the MSNs to study their behavior inside the cells, and the time was optimized from previous studies with this cell types [38]. Each experiment was performed in triplicate, and representative results were shown per group tested. Cell counting was performed using trypan-blue staining (0.4%, Invitrogen™), which quantifies membrane-compromised cells, in a Countess TM cell counter where cell number and viability are measured by exclusion on a disposable slide with a cell-counting chamber.

Viability and Cytotoxicity
Cytotoxicity was measured by a quantification of viability at 24/48 h using Alamar Blue ® reagent (Invitrogen™, Groningen, The Netherlands) (according to the manufac-  [36]. Cellular metabolic activity was quantified by the conversion of resazurin to resorufin by measuring its fluorescence at λ EX 560 nm/λ EM 590 nm on a Synergy 2 luminescence multimode microplate reader (BioTek ® , Winooski, VT, USA) after 2 h of cell internalization.

Cell Uptake
Flow-cytometry measurements were performed using flow cytometry (IS conditions) on a FACScan (Becton, Dickinson and Company, Mountain View, CA, USA) at an excitation wavelength of 488 nm, measuring at 530 nm, and the activator was set to the green fluorescence channel (FL1) [40]. For statistical significance, at least 10,000 cells in each sample were analyzed, and the mean fluorescence emitted by cells that took up the fluorescein-labelled MSN@PEI nanocarrier was used (25, 50 and 100 µg/mL). Trypan blue was used to eliminate extracellular fluorescence. Images obtained to confirm internalization were taken using an optical microscope at 20× magnification.

Mineralization
Osteoblastic-mineralization studies were carried out by performing an alizarin-red staining after 15 days. Calcium deposits formed after treating cells for 2 h with 0.05 nM PTN and after 100 µg/mL MSN@PEI-PTN were quantified. In the present mineralization assay, alizarin-red reagent (0.136 g, 40 mM pH 4.2) was used as a dye with an affinity to the calcium ion, an ion secreted by hMSCs upon differentiation [38]. This dye was eluted with 10% p/v cetylperidinium chloride reagent in 10 mM sodium phosphate buffer of pH 7. All reagents were obtained from Merck, Spain. Finally, absorbance was measured at 620 nm in a Synergy 2 multimode luminescence microplate reader (BioTek ® ) to quantify the mineralization produced.

Gene Expression
Both cell lines were cultured for 5 days in the presence or absence of different nanosystems for differential gene-expression studies. The gene expression of the osteoblasticdifferentiation markers, Alkaline Phosphatase (ALP) and Runx2, were quantified using real-time PCR, QuantStudio5 equipment, and a previously described protocol (Applied Biosystem-Thermo Scientific, Foster City, CA, USA) [36]. TaqMan MGB probes were obtained using Assay-by-Design SM (Applied Biosystems). For each sample and using the given cycle-threshold (Ct) value, mRNA copy numbers were calculated and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rRNA (a housekeeping gene) was amplified in parallel with the tested genes. The number of amplification steps required to reach an arbitrary Ct was computed. The relative gene expression was represented by 2 −∆∆Ct , where ∆∆Ct = ∆C target gene − ∆Ct GAPDH . The fold change for the treatment was defined as the relative expression compared to the control GAPDH expression, calculated as 2 −∆∆Ct , where ∆∆Ct = ∆C treatment − ∆C control .

Statistical Analysis
The results presented below are expressed as mean ± standard error of the mean (SEM). Statistical analyses of differences between groups were performed using the Student's t-test. All values of p < 0.05 were considered significant.

Results and Discussion
The limitations in the current treatments of osteoporosis have made it necessary to search for different alternatives [6,7]. In this sense, nanotechnology might provide advanced alternatives to solve these problems [24,[26][27][28]31]. To this end, a novel nanosystem based on MSNs loaded with a pleiotrophin osteogenic peptide and coated with PEI polymer, which improves the internalization of the nanocarrier, has been developed and evaluated here in two bone-related cell lines. Internalization, viability, mineralization, and differentiation gene-expression studies were performed to show the efficacy of this nanosystem in mouse pre-osteoblastic MC3T3-E1 cells and human Mesenchymal Stem Cells (hMSC) isolated from normal adult human bone marrow extracted from bilateral punctures of the posterior iliac crests of healthy volunteers [38,39].

Synthesis and Physico-Chemical Characterization of Nanosystems
Mesoporous silica nanoparticles were produced following a modification of the Stöber method in which a surfactant was employed as a structure-directing agent to produce the mesostructured silica. Nanoparticles with mesoporous structure and a spherical shape of ca. 80-120 nm in diameter, measured by DLS, were obtained. The MSNs were coated with PEI to ensure the PTN loading and favor cellular internalization ( Figure 1A).
The correct synthesis of MSNs and the subsequent modification of their surface were confirmed through several characterization techniques. Transmission-electron microscopy of the MSNs revealed the small size and mesoporous structure of the particles ( Figure 1B). The surface properties of the MSNs were analyzed using a N 2 isotherm according to a Brunauer-Emmett-Teller (BET) model with an area of 1.150 ± 10 m 2 /g, a pore volume of 1.12 cm 3 /g, and a pore size that was adjusted to 3.94 nm ( Figure 1D), and obtained by a method based on density functional theory (DFT). The diffraction maxima corresponding to the 100, 110, and 200 planes were analyzed, confirming the hexagonal mesostructured of the pores, which is typical of MCM-41 materials (Figure 1) [20][21][22][23]. These data confirm that the external surface of the particles was successfully grafted with the polymer.
The nanoparticles were functionalized with phosphonate groups, providing the strong negative charge needed to coat them afterwards with the cationic PEI polymer. The functionalization of the nanoparticles with PEI was confirmed by several characterization techniques. In this regard, the correct polymer coating, which was not present on unmodified MSNs, was confirmed through the phosphotungstic-acid-stained layer covering the surface of PEI-functionalized nanoparticles (MSN@PEI), and it can be appreciated by TEM in Figure 1B The Z-potential measurements of the particles dispersed in water confirmed the successful functionalization of MSNs with PEI. As previously mentioned, the MSNs were initially functionalized with phosphonate groups, inducing a higher negative charge (−20 mV) than MSNs without phosphonate groups (−5 mV), which are required to efficiently coat with the polymer. In this sense, there was a severe change from negative phosphonate functionalized MSNs (−20 mV) to positive (+26 mV) in PEI-coated MSNs (Figures 1 and  2). The hydrodynamic size of the MSNs was evaluated using dynamic light scattering, showing that their monodispersity was unaffected by the functionalizing process. The amount of PEI grafted onto the MSN surface was quantified using thermal analyses, as observed in Figure 2. All these data confirmed that the external surface of the MSNs was successfully grafted with the PEI polymer.
Recombinant human-protein pleiotrophin (PTN) loading was carried out in a 1:4-10 −4 MSN@PEI-PTN ratio, where, for each mg of MSN@PEI, there would be 0.4 μg of PTN. The protein loading was confirmed using FTIR to confirm the presence of typical C=O vibration bands from the PTN (Figure 2), in addition to the DLS results, where a slight increase of the hydrodynamic size was observed because of the PTN loading to the surface of the nanoparticles and the Z-potential values, which shifted from +26 mV of unloaded carriers to 13.5 mV in PTN-loaded nanoparticles. The charge reduction might be ascribed to the biomolecule presence on the surface of the developed MSNs. Moreover, the thermogravimetric analysis quantified 17% by the weight of PEI decorating the surface of the nanoparticles, and 30% of PTN proteins loaded in the nanocarrier system. The Z-potential measurements of the particles dispersed in water confirmed the successful functionalization of MSNs with PEI. As previously mentioned, the MSNs were initially functionalized with phosphonate groups, inducing a higher negative charge (−20 mV) than MSNs without phosphonate groups (−5 mV), which are required to efficiently coat with the polymer. In this sense, there was a severe change from negative phosphonate functionalized MSNs (−20 mV) to positive (+26 mV) in PEI-coated MSNs (Figures 1 and 2). The hydrodynamic size of the MSNs was evaluated using dynamic light scattering, showing that their monodispersity was unaffected by the functionalizing process. The amount of PEI grafted onto the MSN surface was quantified using thermal analyses, as observed in Figure 2. All these data confirmed that the external surface of the MSNs was successfully grafted with the PEI polymer.
Recombinant human-protein pleiotrophin (PTN) loading was carried out in a 1:4-10 −4 MSN@PEI-PTN ratio, where, for each mg of MSN@PEI, there would be 0.4 µg of PTN. The protein loading was confirmed using FTIR to confirm the presence of typical C=O vibration bands from the PTN (Figure 2), in addition to the DLS results, where a slight increase of the hydrodynamic size was observed because of the PTN loading to the surface of the nanoparticles and the Z-potential values, which shifted from +26 mV of unloaded carriers to 13.5 mV in PTN-loaded nanoparticles. The charge reduction might be ascribed to the biomolecule presence on the surface of the developed MSNs. Moreover, the thermogravimetric analysis quantified 17% by the weight of PEI decorating the surface of the nanoparticles, and 30% of PTN proteins loaded in the nanocarrier system.
In the present study, we loaded MSN@PEI with PTN in an attempt to develop an alternative treatment for osteoporosis by improving bone regeneration and increasing bone-cell viability, mineralization, and differentiation. This research is a proof-of-concept for this novel platform. In this sense, we have preliminarily addressed this hypothesis using an in vitro study with two bone-related type cells, MC3T3-E1 cells and hMSCs.

Mouse Pre-Osteoblastic MC3T3-E1 Cells
The first in vitro study to be carried out analyzed the cell uptake of nanosystems, with and without PEI coating, in order to find out whether the presence of the polymer could increase internalization after 2 h of MSN incubation. Flow-cytometry studies showed a significant increase in the internalization rate in mouse pre-osteoblastic MC3T3-E1 cells in a dose-dependant manner in both MSN and MSN@PEI nanosystems (Figure 3), with MSN@PEI at 100 μg/mL being the most effective. As shown in Figure 3, Rhodamine-B-labeled MSN@PEI (in red) were found in the cytoplasm, mostly around the nucleus that was stained blue with DAPI. This perinuclear location might be explained by the positive charge of the loaded MSNs ( Figure 2B) and the negatively charged membrane of the nuclei. Furthermore, as discussed above, the present internalization results agree that coating with cationic polymers such as PEI increased the internalization of MSNs and endosomal escape due to the proton-sponge effect [31,32,36]
In the present study, we loaded MSN@PEI with PTN in an attempt to develop an alternative treatment for osteoporosis by improving bone regeneration and increasing bone-cell viability, mineralization, and differentiation. This research is a proof-of-concept for this novel platform. In this sense, we have preliminarily addressed this hypothesis using an in vitro study with two bone-related type cells, MC3T3-E1 cells and hMSCs.

Mouse Pre-Osteoblastic MC3T3-E1 Cells
The first in vitro study to be carried out analyzed the cell uptake of nanosystems, with and without PEI coating, in order to find out whether the presence of the polymer could increase internalization after 2 h of MSN incubation. Flow-cytometry studies showed a significant increase in the internalization rate in mouse pre-osteoblastic MC3T3-E1 cells in a dose-dependant manner in both MSN and MSN@PEI nanosystems (Figure 3), with MSN@PEI at 100 µg/mL being the most effective. As shown in Figure 3, Rhodamine-Blabeled MSN@PEI (in red) were found in the cytoplasm, mostly around the nucleus that was stained blue with DAPI. This perinuclear location might be explained by the positive charge of the loaded MSNs ( Figure 2B) and the negatively charged membrane of the nuclei. Furthermore, as discussed above, the present internalization results agree that coating with cationic polymers such as PEI increased the internalization of MSNs and endosomal escape due to the proton-sponge effect [31,32,36]  The next step in the biological evaluation of this nanocarrier was to show that none of the nanocarriers are toxic in the presence of different cell cultures. As is well-known, some cationic polymers, including PEI, can be toxic in different cell lines. The PEI used in this work was 5KD, which has been previously demonstrated to be non-toxic by Maria Vallet's research group and other groups [33,34,36,40]. In this sense, viability results in mouse pre-osteoblastic cells (measured by Alamar Blue ® ) indicated that none of the nanosystems studied the affected cell viability at 24 and 48 h of cell culture, in the presence or absence of PEI polymer (Figure 4). The increase of the nanocarrier concentration slightly reduced the viability, as previously shown in the literature [31,32,40].
After internalization and cytotoxic evaluation of the here-developed nanoplatform, a concentration of 100 μg/mL of the MSN@PEI nanosystem was selected as the optimal concentration for the rest of the experiments. Very interesting results were observed when evaluating PTN-loaded MSNs with these cells. MSN@PEI-PTN nanocarrier significantly increased viability in comparison with free PTN at a 0.05 nM concentration, especially at 24 h, confirming the lack of cytotoxicity of the nanocarrier here-developed for the first time ( Figure 4). These results demonstrate that PTN can be efficiently loaded and released from MSNs, producing an intense increase in bone-related cell viability even higher than that of free PTN. This is an important point since, as a growth factor in bone tissue, PTN must be administered in a localized manner as a part of ectopic osteogenic growth. The encapsulation of this biomolecule in the nanoplatform here-developed would ensure the local delivery of the PTN where it would produce a beneficial effect in the bone-tissue environment. Once there, the PTN release would increase bone-related cell viability and, therefore, bone regeneration.
In addition to improving the viability of pre-osteoblasts, the present nanosystems also significantly increase the mineralization and differentiation of these cells in carrying out their reparative function in situations of a loss of bone mass or function. For this reason, is important to study whether the complete MSN@PEI nanosystem loaded with PTN is capable of increasing calcium deposits (protein) and the gene expression of classic bone markers involved in osteoblast differentiation. It is important to note that the experiments carried out were performed in the absence of an osteogenic differentiation-promoting medium, which gives even more validity to the results obtained in this study related to osteoblastic differentiation and mineralization. Most studies involving the measurement of The next step in the biological evaluation of this nanocarrier was to show that none of the nanocarriers are toxic in the presence of different cell cultures. As is well-known, some cationic polymers, including PEI, can be toxic in different cell lines. The PEI used in this work was 5KD, which has been previously demonstrated to be non-toxic by Maria Vallet's research group and other groups [33,34,36,40]. In this sense, viability results in mouse pre-osteoblastic cells (measured by Alamar Blue ® ) indicated that none of the nanosystems studied the affected cell viability at 24 and 48 h of cell culture, in the presence or absence of PEI polymer (Figure 4). The increase of the nanocarrier concentration slightly reduced the viability, as previously shown in the literature [31,32,40].
After internalization and cytotoxic evaluation of the here-developed nanoplatform, a concentration of 100 µg/mL of the MSN@PEI nanosystem was selected as the optimal concentration for the rest of the experiments. Very interesting results were observed when evaluating PTN-loaded MSNs with these cells. MSN@PEI-PTN nanocarrier significantly increased viability in comparison with free PTN at a 0.05 nM concentration, especially at 24 h, confirming the lack of cytotoxicity of the nanocarrier here-developed for the first time ( Figure 4). These results demonstrate that PTN can be efficiently loaded and released from MSNs, producing an intense increase in bone-related cell viability even higher than that of free PTN. This is an important point since, as a growth factor in bone tissue, PTN must be administered in a localized manner as a part of ectopic osteogenic growth. The encapsulation of this biomolecule in the nanoplatform here-developed would ensure the local delivery of the PTN where it would produce a beneficial effect in the bone-tissue environment. Once there, the PTN release would increase bone-related cell viability and, therefore, bone regeneration.
In addition to improving the viability of pre-osteoblasts, the present nanosystems also significantly increase the mineralization and differentiation of these cells in carrying out their reparative function in situations of a loss of bone mass or function. For this reason, is important to study whether the complete MSN@PEI nanosystem loaded with PTN is capable of increasing calcium deposits (protein) and the gene expression of classic bone markers involved in osteoblast differentiation. It is important to note that the experiments carried out were performed in the absence of an osteogenic differentiation-promoting medium, which gives even more validity to the results obtained in this study related to osteoblastic differentiation and mineralization. Most studies involving the measurement of these parameters used β-glycerolphosphate and ascorbic acid to force the mineralization to more differentiated phenotypes, either in presence or absence of different types of biomaterials [41][42][43].
Pharmaceutics 2023, 15, x FOR PEER REVIEW 10 of 16 these parameters used β-glycerolphosphate and ascorbic acid to force the mineralization to more differentiated phenotypes, either in presence or absence of different types of biomaterials [41][42][43]. The differentiation assays revealed an increase in the mineralization of cells exposed to MSN@PEI compared to free PTN ( Figure 5), confirming that the designed MSN@PEI-PTN system was effective for increasing the calcium deposits and could improve bioavailability in bone tissue. Although both PTN alone and PTN loaded into the nanocarrier significantly increased mineralization in these cells, which is also positive for the reasons mentioned above, no significant differences were observed between them. Besides the positive effect of our nanoparticles on osteoblast differentiation, we also evaluated the expression of two genes, Runx2 and ALP, that are indicative of an early bone-regeneration process. In this sense, the complete nanocarrier with PEI and PTN significantly increased the expression of Runx2 and ALP over the values of PTN. Again, the use of MSNs to deliver PTN has demonstrated better results than free PTN in triggering the bone-regeneration process through the expression of these genes. Although both free PTN and PTN loaded into the nanocarrier significantly increased the mineralization of these cells, the real effect was observed in the case of gene expression, where the complete nanosystem with PEI and PTN significantly increased the expression of Runx2 and ALP over the values of free PTN. These results support previous data regarding the use of PTN in a biomimetic scaffold [44]. The differentiation assays revealed an increase in the mineralization of cells exposed to MSN@PEI compared to free PTN ( Figure 5), confirming that the designed MSN@PEI-PTN system was effective for increasing the calcium deposits and could improve bioavailability in bone tissue. Although both PTN alone and PTN loaded into the nanocarrier significantly increased mineralization in these cells, which is also positive for the reasons mentioned above, no significant differences were observed between them. Besides the positive effect of our nanoparticles on osteoblast differentiation, we also evaluated the expression of two genes, Runx2 and ALP, that are indicative of an early bone-regeneration process. In this sense, the complete nanocarrier with PEI and PTN significantly increased the expression of Runx2 and ALP over the values of PTN. Again, the use of MSNs to deliver PTN has demonstrated better results than free PTN in triggering the bone-regeneration process through the expression of these genes. Although both free PTN and PTN loaded into the nanocarrier significantly increased the mineralization of these cells, the real effect was observed in the case of gene expression, where the complete nanosystem with PEI and PTN significantly increased the expression of Runx2 and ALP over the values of free PTN. These results support previous data regarding the use of PTN in a biomimetic scaffold [44].

Human Mesenchymal Stem Cells (hMSCs)
Based on the above results, which demonstrated a positive effect of PTN loaded into the MSN@PEI nanosystem on the viability, mineralization, and differentiation of mouse pre-osteoblastic cells, an additional human cell line was selected to confirm these positive findings. Therefore, similar studies were carried out with human bone marrow-derived mesenchymal cells. Those cells were selected for the present study because of their human source and because they are known to play an important role in bone-regeneration processes [38].
The obtained cell-internalization results were in agreement with those obtained for MC3T3-E1 cells. Cell-uptake studies by flow cytometry indicated a significant increase in the internalization rate in hMSCs in a dose-dependent manner in the presence of MSN@PEI nanosystems ( Figure 6) at 25, 50, and 100 μg/mL concentrations, with MSN@PEI at 100 μg/mL being the most effective. In this sense, Figure 6 shows how Fluorescein-labeled MSN@PEI (in green) are located in the cytoplasms (in red) of hMSCs, as initially hypothesized here.

Human Mesenchymal Stem Cells (hMSCs)
Based on the above results, which demonstrated a positive effect of PTN loaded into the MSN@PEI nanosystem on the viability, mineralization, and differentiation of mouse preosteoblastic cells, an additional human cell line was selected to confirm these positive findings. Therefore, similar studies were carried out with human bone marrow-derived mesenchymal cells. Those cells were selected for the present study because of their human source and because they are known to play an important role in bone-regeneration processes [38].
The obtained cell-internalization results were in agreement with those obtained for MC3T3-E1 cells. Cell-uptake studies by flow cytometry indicated a significant increase in the internalization rate in hMSCs in a dose-dependent manner in the presence of MSN@PEI nanosystems ( Figure 6) at 25, 50, and 100 µg/mL concentrations, with MSN@PEI at 100 µg/mL being the most effective. In this sense, Figure 6 shows how Fluorescein-labeled MSN@PEI (in green) are located in the cytoplasms (in red) of hMSCs, as initially hypothesized here.

Human Mesenchymal Stem Cells (hMSCs)
Based on the above results, which demonstrated a positive effect of PTN loaded the MSN@PEI nanosystem on the viability, mineralization, and differentiation of m pre-osteoblastic cells, an additional human cell line was selected to confirm these pos findings. Therefore, similar studies were carried out with human bone marrow-der mesenchymal cells. Those cells were selected for the present study because of their hu source and because they are known to play an important role in bone-regeneration cesses [38].
The obtained cell-internalization results were in agreement with those obtaine MC3T3-E1 cells. Cell-uptake studies by flow cytometry indicated a significant increa the internalization rate in hMSCs in a dose-dependent manner in the presenc MSN@PEI nanosystems ( Figure 6) at 25, 50, and 100 μg/mL concentrations, MSN@PEI at 100 μg/mL being the most effective. In this sense, Figure 6 shows how F rescein-labeled MSN@PEI (in green) are located in the cytoplasms (in red) of hMSC initially hypothesized here. The viability results of hMSCS were slightly different from those obtained in MC3T3-E1 cells. The best viability of hMSCs cells was observed when using MSN@PEI, with the best concentration being 100 µg/mL MSNs (Figure 7). However, a PEI coating slightly affected viability, although this effect disappeared over time. This fact could be explained by the mesenchymal nature of the cells, where the effects tend to be stronger due to their greater reactivity to stimuli. However, the presence of PEI at the surface of the MSNs did not drastically affect the cell viability in comparison with the hMSC control ( Figure 7). As was the case with the MC3T3-E1 cells, the MSN@PEI nanocarrier at a concentration of 100 µg/mL was chosen for the rest of the experiments (Figures 7 and 8) based on the internalization and viability results.
Very impressive results in human mesenchymal-cell viability were observed when loading PTN into the nanosystem. MSN@PEI-PTN nanosystems significantly increased viability over free PTN at a 0.05 nM concentration. A remarkable increase was observed in the first 24 h, indicating that the protein might be released. Additionally, loading PTN to our nanoplatform almost doubled hMSC viability, confirming the very positive effect of the nanocarrier here-designed in human bone cells. The viability results of hMSCS were slightly different from those obtained in MC3T3-E1 cells. The best viability of hMSCs cells was observed when using MSN@PEI, with the best concentration being 100μg/mL MSNs (Figure 7). However, a PEI coating slightly affected viability, although this effect disappeared over time. This fact could be explained by the mesenchymal nature of the cells, where the effects tend to be stronger due to their greater reactivity to stimuli. However, the presence of PEI at the surface of the MSNs did not drastically affect the cell viability in comparison with the hMSC control ( Figure 7). As was the case with the MC3T3-E1 cells, the MSN@PEI nanocarrier at a concentration of 100 μg/mL was chosen for the rest of the experiments (Figures 7 and 8) based on the internalization and viability results.
Very impressive results in human mesenchymal-cell viability were observed when loading PTN into the nanosystem. MSN@PEI-PTN nanosystems significantly increased viability over free PTN at a 0.05 nM concentration. A remarkable increase was observed in the first 24 h, indicating that the protein might be released. Additionally, loading PTN to our nanoplatform almost doubled hMSC viability, confirming the very positive effect of the nanocarrier here-designed in human bone cells.   The differentiation assays revealed an increase in the mineralization of hMSCs exposed to MSN@PEI-PTN and PTN, as previously observed in pre-osteoblastic cells. These results confirmed that the MSN@PEI-PTN system was as effective as the free protein alone (Figure 8). The consistency of this effect with results similar to those obtained in MC3T3-E1 cells confirms the efficiency of the nanoplatform proposed here independently of the cell evaluated. In addition, the complete nanocarrier with PEI and PTN improved the gene expression of Runx2 and ALP compared to that in free PTN. As observed in the mineralization, the results obtained in the gene expression of these osteoblastic markers in hMSCs were in agreement with those of mouse pre-osteoblastic cells, which confirms again that the nanosystem proposed here would trigger bone-tissue regeneration regardless of the cells line evaluated.
the presence of 0.05 nM exogenous PTN alone at 24 and 48 h of cell culture. Data are mean ± SEM of three independent experiments performed in triplicate. * p < 0.05 vs. hMSC and MSN@PEI 100 μg/mL; # p < 0.01 vs. PTN 0.05 nM and all groups.
The differentiation assays revealed an increase in the mineralization of hMSCs exposed to MSN@PEI-PTN and PTN, as previously observed in pre-osteoblastic cells. These results confirmed that the MSN@PEI-PTN system was as effective as the free protein alone (Figure 8). The consistency of this effect with results similar to those obtained in MC3T3-E1 cells confirms the efficiency of the nanoplatform proposed here independently of the cell evaluated. In addition, the complete nanocarrier with PEI and PTN improved the gene expression of Runx2 and ALP compared to that in free PTN. As observed in the mineralization, the results obtained in the gene expression of these osteoblastic markers in hMSCs were in agreement with those of mouse pre-osteoblastic cells, which confirms again that the nanosystem proposed here would trigger bone-tissue regeneration regardless of the cells line evaluated. In addition, it is significant to note that the expression levels were higher than in the case of pre-osteoblastic cells. These results are in agreement with previous data that demonstrate that PTN transactivates endothelial-growth-factor receptor-activating Erk and Akt pathways in the primary osteoblast, which leads to an increase in ALP activity [45]. However, loading PTN into MSNs has doubled that positive effect on early boneregeneration gene expression, which means that the platform developed here for the first time could be part of a new and effective treatment for bone regeneration.

Conclusions
The present proof-of-concept showed, for the first time, that PTN-protein loaded into mesoporous silica nanoparticles improved the viability and the osteogenic and differentiating actions in the absence of osteogenic differentiation-promoting medium in different cell lines. The here-developed nanoplatform was evaluated in two bone related cell lines, pre-osteoblastic cells and human mesenchymal stem cells, showing that the promising results in terms of viability and bone regeneration were independent of the in vitro model employed. The synthesis, functionalization, and PTN loading have been optimized to develop an effective pleiotrophin nanocarrier for the first time. The use of this novel nanoplatform increased the cell viability in comparison with free PTN and, more importantly, doubled the expression of certain genes indicative of the early bone-regeneration process. In addition, it is significant to note that the expression levels were higher than in the case of pre-osteoblastic cells. These results are in agreement with previous data that demonstrate that PTN transactivates endothelial-growth-factor receptor-activating Erk and Akt pathways in the primary osteoblast, which leads to an increase in ALP activity [45]. However, loading PTN into MSNs has doubled that positive effect on early bone-regeneration gene expression, which means that the platform developed here for the first time could be part of a new and effective treatment for bone regeneration.

Conclusions
The present proof-of-concept showed, for the first time, that PTN-protein loaded into mesoporous silica nanoparticles improved the viability and the osteogenic and differentiating actions in the absence of osteogenic differentiation-promoting medium in different cell lines. The here-developed nanoplatform was evaluated in two bone related cell lines, preosteoblastic cells and human mesenchymal stem cells, showing that the promising results in terms of viability and bone regeneration were independent of the in vitro model employed. The synthesis, functionalization, and PTN loading have been optimized to develop an effective pleiotrophin nanocarrier for the first time. The use of this novel nanoplatform increased the cell viability in comparison with free PTN and, more importantly, doubled the expression of certain genes indicative of the early bone-regeneration process. These findings could be of great importance for future developments of nanosystems for the potential treatment of osteoporosis or other bone-related diseases where fast and effective bone regeneration might be required.